The objective of this research proposal is to understand the biosynthetic pathways leading to inhibin secretion from ovarian granulosa cells. The most recognized action of inhibin is to suppress FSH production and secretion by the pituitary gonadotrope in vitro and in vivo. Inhibin levels are regulated during the reproductive cycle to ensure normal FSH-dependent follicle recruitment. Little is known regarding the cellular pathways leading to inhibin biosynthesis, yet, it is the thesis of this grant renewal that these pathways are responsible for the accurate production of this key ovarian feedback hormone and are vital to normal reproductive function. Inhibin A and inhibin B are produced by the granulosa cell of the ovary, however, the secretion patterns for these ligands are distinct, suggesting an underlying control over biosynthesis and release that has not been previously investigated. In addition, the precise mechanism by which many granulosa cell products are targeted toward the antral fluid or the vascular theca cell compartment is not well understood. Our preliminary studies have identified differential secretion patterns of inhibin A and inhibin B during the rat reproductive cycle, described differential compartmentalization of inhibin subunit protein in granulosa cells of developing follicles, and revealed that the biosynthetic processing of inhibin isoforms is regulated. These preliminary studies and the results of the past five years of work suggest that the cellular machinery that regulates inhibin biosynthesis and processing is important to inhibin action and must be investigated in order to more completely understand the function of this ligand in women and men. We hypothesize that inhibin bioactivity relies on post-translational modification including N-linked glycosylation, protein specific routing signals contained in pro-hormone domains, and bioprocessing enzymes that convert bioinactive pro-ligand into mature, bioactive inhibin. There are three interrelated experimental aims in this proposal that will address the central hypothesis and its tenets. These studies are expected to provide insight into the control of inhibin synthesis and release and contribute to a more complete understanding of normal fertility and the mechanisms that underlie fertilityrelated diseases in women resulting from inappropriate hormone action.